Voltage-dependent calcium channels have been linked to physiological processes such as neurotransmitter release, secretion of hormones, muscle contraction, and regulation of gene transcription. A functional channel requires at least three subunits, including the α1, α2δand β subunits. The channel may also contain a γ subunit. There are several known types of voltage-dependent calcium channels that have been defined based on their electrophysiological characteristics and pharmacological properties. These types are L-, N-, P/Q-, R-, and T-type. Each type is primarily defined by its channel composition. The type of α1 subunit contained in the channel determines whether the channel is an L-, N-, P/Q-, R-, or T-type channel. The activity of the α1 subunit is modulated by the α2δ and β subunits. Channel activity may be further modulated by a fourth subunit, γ.
Molecular biological techniques have allowed elucidation of the mechanism of voltage-dependent calcium channel action. Genes for each of the subunits have been isolated and cloned. There are currently nine known genes encoding for different α1 subunits. The a1 subunit forms the pore which calcium ions flow through. The α1 subunit contains the voltage sensor and is also responsible for the binding specificity of certain drugs or toxin that may be associated with the channel type. Channel current through the α1 pore may be modulated by association of the β, γ, or α2δ subunit. There are four known genes for the intracellular β subunit that may be differentially spliced. There are two known genes for the transmembraneγ subunit, one in skeletal muscle and a novel gene expressed in the brain. Only one isoform of α2δ was initially identified. Recently, however, two new α2δ genes were identified, α2δ2 and α2δ3. These genes have 55.6 and 30.3% homology with the original α2δ1 gene (Klugbauer, et al., J Neuroscience 1999;19(2):684–691). The α2 and δ proteins are expressed by the same gene. The protein product is post-translationally cleaved, and the final. α2 and δ proteins are linked by disulfide bonds. The transmembrane δ protein secures the α2 protein to the cell membrane.
Studies have shown that the α2δ1 subunit contains a binding site for the anticonvulsant drug, gabapentin [1-(aminomethyl)cyclohexane acetic acid] (Gee, et al., J. Biol. Chem. 1996;271(10):5768–5776). Gabapentin is a γ-aminobutyric acid (GABA) analogue. Gabapentin is effective in the treatment of epilepsy and in decreasing seizure frequency in both animal models and in human patients. The precise mechanism of action of gabapentin remains unclear. Recent experiments have shown that gabapentin also binds to the α2δ2 subunit.
Functional channels may be formed by expression of the calcium channel subunits in a cell. This technique is advantageous in determining the effects of various molecules on channel action. U.S. Pat. No. 5,712,158 and U.S. Pat. No. 5,770,447 describe a stable cell line that is useful for investigating gabapentin binding properties to calcium channel subunits. This cell line expresses the β subunit and the original α2δ subunit (now referred to as α2δ1) at high levels. Transfecting the cells with any α1 subunit results in the formation of functional calcium channels which can be used to evaluate the binding of gabapentin and gabapentin-related compounds.
It is the object of this invention to provide a new cell line that stably expresses a calcium channel α2δ2 subunit. It is a further object of this invention to describe α2δ2 subtype-specific binding of gabapentin, analogues of gabapentin, pregabalin, analogues of pregabalin, and 3-alkyl derivatives of GABA.